MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells.

BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.

MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells

Background: Mast cell (MC) degranulation via activation of the Mas-related G protein–coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. Methods: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. Results: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. Conclusion: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing (AU)

Antecedentes: La desgranulación de los mastocitos (MC) a través de la activación del receptor X2 acoplado a proteína G relacionada con Mas (MRGPRX2) se considera clave para la hipersensibilidad inmediata a fármacos. Sin embargo, los datos en humanos se limitan a observaciones en líneas celulares específicas. Objetivo: Estudiar la utilidad del silenciamiento de MRGPRX2 en MC humanos para conocer mejor la vía MRGPRX2 en la hipersensibilidad inmediata a fármacos. Métodos Los MC se cultivaron a partir de células progenitoras CD34+ obtenidas de sangre periférica (PBCMC) y se incubaron con la sustancia P como control positivo, rocuronio, moxifloxacina, morfina o amoxicilina. El inmunofenotipaje de las células incluyó análisis por citometría de flujo y microscopia de la expresión de CD117, CD203c y MRGPRX2. El calcio intracelular se midió usando Fluo-4. La desgranulación se analizó por cuantificación de la expresión de CD63. Para el silenciamiento de MRGPRX2, los MC se electroporaron con ARN silente del sustrato Dicer. Resultados: La incubación de MC con sustancia P, morfina y moxifloxacina provocó el aumento de los niveles de calcio intracelular y desencadenó la desgranulación de MC. En el caso de la desgranulación provocada por los fármacos, ésta se eliminó casi por completo mediante el silenciamiento selectivo de MRGPRX2. A pesar del aumento del calcio intracelular en las células MRGPRX2+, la incubación con concentraciones no tóxicas de rocuronio no produce la desgranulación de los PBCMC, mientras que la amoxicilina no tiene efecto sobre los PBCMC. Conclusión: El uso del silenciamiento de MRGPRX2 en MC humanos puede proporcionar información importante sobre el papel de MRGPRX2 en la patogénesis de la hipersensibilidad inmediata a fármacos. Como la inducción de señales de calcio no se traduce necesariamente en una respuesta secretora, parece más significativa la medición de la reacción de desgranulación en el contexto de las pruebas a fármacos (AU)

Immediate moxifloxacin hypersensitivity: Is there more than currently meets the eye?

Immediate drug hypersensitivity reactions (IDHR) to moxifloxacin constitute a pathomechanistic conundrum and a diagnostic challenge. Our objective was to study whether simultaneous phenotyping and quantification of histamine release might add to our knowledge about the basophil activation properties of moxifloxacin and constitute a reliable diagnostic aid. Fifteen patients with an IDHR to moxifloxacin and nine moxifloxacin challenged controls were selected. All had a basophil activation test (BAT) with moxifloxacin. Flow cytometric analysis of basophil responses implied labeling for CD63, CD203c, and intracellular histamine. Unlike tolerant challenged controls, basophilic upregulation of CD203c in response to moxifloxacin was observed in seven of 15 patients. Only two of these seven patients demonstrated appearance of CD63 and release of histamine. In the remainder eight patients, no basophil responses were demonstrable. In conclusion, immediate hypersensitivity to moxifloxacin might involve mechanisms difficult to capture by traditional CD63-/CD203c-based BAT. Deciphering the complexity of quinolone IDHR seems mandatory.

In Vitro Diagnosis of Immediate Drug Hypersensitivity Anno 2017: Potentials and Limitations.

BACKGROUND: For most physicians, quantification of drug-specific immunoglobulin E (drug-sIgE) antibodies constitutes the primary in vitro measure to document immediate drug hypersensitivity reactions (IDHR). Unfortunately, this is often insufficient to correctly identify patients with IgE-mediated IDHR and impossible for non-IgE-mediated IDHR that result from alternative routes of basophil and mast cell activation. In these difficult cases, diagnosis might benefit from cellular tests such as basophil activation tests (BAT). AIM: The aim was to review the potential and limitations of quantification of sIgE and BAT in diagnosing IDHR. The utility of quantification of serum tryptase is discussed. METHODS: A literature search was conducted using the key words allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry, specific IgE antibodies; this was complemented by the authors' own experience. RESULTS: The drugs that have been most studied with both techniques are ß-lactam antibiotics and curarizing neuromuscular blocking agents (NMBA). For sIgE morphine, data are available on the value of this test as a biomarker for sensitization to substituted ammonium structures that constitute the major epitope of NMBA, especially rocuronium and suxamethonium. For the BAT, there are also data on non-steroidal anti-inflammatory drugs (NSAIDs) and iodinated radiocontrast media. For ß-lactam antibiotics, sensitivity and specificity of sIgE varies between 0 and 85% and 52 and 100%, respectively. For NMBA, sensitivity and specificity varies between 38.5 and 92% and 85.7 and 100%, respectively. Specific IgE to morphine should not be used in isolation to diagnose IDHR to NMBA nor opiates. For the BAT, sensitivity generally varies between 50 and 60%, whereas specificity attains 80%, except for quinolones and NSAIDs. CONCLUSIONS: Although drug-sIgE assays and BAT can provide useful information in the diagnosis of IDHR, their predictive value is not absolute. Large-scale collaborative studies are mandatory to harmonize and optimize test protocols and to establish drug-specific decision thresholds.

Phenotypic and functional characterization of in vitro cultured human mast cells.

BACKGROUND: Mast cell progenitor cells, derived from CD34+ hematopoietic stem cells, enter the circulation and subsequently mucosal or connective tissues where they mature to mast cells. Upon activation, mast cells increase the expression of activation markers, e.g. CD63, and release histamine amongst other mediators. Traditionally, release of these mediators is quantified using assays measuring their extracellular concentration in the supernatant of stimulated cells. METHODS: Human mast cells (HuMC) were cultured from peripheral blood, phenotypically characterized, passively sensitized with allogenic IgE antibodies and finally stimulated by anti-IgE that crosslinks IgE/FcεRI complexes. Alterations in the number of cells positive for CD63 and release of histamine were quantified simultaneously by flow cytometry. RESULTS: In culture, two distinct CD45+ cell populations were identified: CD117+ CD203c+hi and CD117- CD203c+low cells. Both populations showed positivity for FcεRI, tryptase and chymase, and contained histamine. Activation resulted in a significant increase of cells positive for CD63+ up to 21% (range: 11-39) for CD117+ CD203c+hi cells (P = 0.005), and 27% (18-55) CD63+ for CD117- CD203c+low cells (P = 0.02). Baseline histamine content was higher for CD117+ CD203c+hi cells than for CD117- CD203c+low cells, respectively 994 (695-6815) Molecules of Equivalent Specific Fluorochrome V500 per cell (MESF-V500/cell) and 797 (629-4978) MESF-V500/cell (P = 0.02). After activation, CD117+ CD203c+hi cells showed significant histamine release of 578 (366-1521) MESF-V500/cell, whilst CD117- CD203c+low cells resulted in 310 (217-366) MESF-V500/cell histamine release. CONCLUSION: This study discloses that culturing HuMC from CD34+ progenitors yields 2 phenotypically distinct cell populations that display a greatly similar response upon cross-linking of IgE/FcεRI complexes. © 2016 International Clinical Cytometry Society.

Shellfish allergens: tropomyosin and beyond.

IgE-mediated shellfish allergy constitutes an important cause of food-related adverse reactions. Shellfish are classified into mollusks and crustaceans, the latter belonging to the class of arthropoda. Among crustaceans, shrimps are the most predominant cause of allergic reactions and thus more extensively studied. Several major and minor allergens have been identified and cloned. Among them, invertebrate tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, and hemocyanin are the most relevant. This review summarizes our current knowledge about these allergens.

Cannabis sativa allergy: looking through the fog.

IgE-mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life-threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross-allergies, mostly for plant foods. This clinical entity, designated as the 'cannabis-fruit/vegetable syndrome', might also imply cross-reactivity with tobacco, natural latex and plant-food-derived alcoholic beverages. Hitherto, these cross-allergies are predominantly reported in Europe and appear mainly to rely upon cross-reactivity between nonspecific lipid transfer proteins or thaumatin-like proteins present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross-allergies remains uncertain.

Quantification of specific IgE antibodies in immediate drug hypersensitivity: More shortcomings than potentials?

BACKGROUND: For many physicians, quantification of serum drug-specific IgE (sIgE) antibodies constitutes the first measure in the diagnostic approach of immediate drug hypersensitivity reactions (IDHR). AIM: To review the accuracy and limitations of the main drug-sIgE tests, especially those that are commercially available. METHODS: A literature search was conducted, using the key-words allergy, diagnosis, drugs, hypersensitivity, specific IgE antibodies; this was complemented by the authors' own experience. RESULTS: The drugs that have mostly been studied appeared to be ß-lactam antibiotics, neuromuscular blocking agents (NMBA) and morphine, the latter as a biomarker for sensitisation to substituted ammonium structures that constitute the major epitope of NMBA. For ß-lactams sensitivity and specificity varied between 0-85% and 52-100%, respectively. For NMBA, sensitivity and specificity varied between 38.5-92% and 92-100%, respectively. With respect to sIgE to morphine it appears this drug to be a sensitive biomarker for sensitisation to rocuronium and suxamethonium but not for atracurium. However, sIgE morphine should not be applied in isolation to diagnose IDHR to NMBA nor opiates. CONCLUSIONS: Although drug-sIgE assay can provide valuable information they should not be performed in isolation to establish correct diagnosis, as their predictive value is not per se absolute. Larger comprehensive studies are urgently required to determine the accuracy of drug-sIgE assays.

Flow-assisted basophil activation tests in immediate drug hypersensitivity: two decades of Antwerp experience.

The last two decades have witnessed that flow-assisted analysis of in vitro-activated basophils can constitute a valuable adjunct in the in vitro diagnostic approach of immediate drug hypersensitivity reactions (IDHR). This article summarises the current experience with the basophil activation test in the diagnosis of IDHR, with particular focus on allergy to curarising neuromuscular blocking agents, antibiotics (ß-lactams and fluoroquinolones), iodinated radiocontrast media and opiates.

New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?

BACKGROUND: Allergy to fruit and vegetables exhibit geographic variation regarding the severity of symptoms and depending on the sensitization profile of the patient. These sensitization profiles and routes remain incompletely understood. Cannabis is a very popular drug and derived from Cannabis sativa, a plant containing lipid transfer proteins (LTP) also known as important allergens in plant and fruit allergies. In this study we sought to elucidate a potential connection between C. sativa allergy and plant food allergies. METHODS: A case-control study involving 21 patients consulting for plant food allergies. Twelve patients were cannabis allergic and 9 had a pollen or latex allergy without cannabis allergy. Testing for cannabis IgE implied measurement of specific IgE, skin testing and basophil activation tests. Allergen component analysis was performed with a microarray technique. RESULTS: Plant food allergy in patients with documented cannabis allergy had more severe reactions than patients without cannabis allergy and frequently implied fruits and vegetables that are not observed in a (birch) pollen-related food syndrome. With the exception of 1 patient with cannabis allergy, all were sensitized to nonspecific (ns)-LTP. CONCLUSION: Our data suggest that illicit cannabis abuse can result in cannabis allergy with sensitization to ns-LTP. This sensitization might result in various plant-food allergies. Additional collaborative studies in different geographical areas are needed to further elucidate on this hypothesis.

IgE-mediated allergy to pholcodine and cross-reactivity to neuromuscular blocking agents: Lessons from flow cytometry.

BACKGROUND: Immunoglubulin E antibody-mediated allergic reactions to opioids are rare and difficult to document correctly. OBJECTIVE: Assessment of the basophil activation test in the diagnosis of IgE-mediated allergy to the antitussive pholcodine and associated sensitizations to neuromuscular blocking agents (NMBA). METHODS: Three patients with a suspected IgE-mediated allergy to pholcodine were investigated using skin tests, quantification of specific IgE, and flow cytometric activation of basophils. RESULTS AND CONCLUSION: Flow cytometric activation of basophils, with simultaneous analysis of CD63 appearance and median histamine content per cell, is the only technique capable to correctly document pholcodine allergy. The negative predictive value of basophil activation tests might help to elucidate on the controversial putative cross-reactivity between pholcodine and NMBA.

Hazelnut allergy: a multi-faced condition with demographic and geographic characteristics.

Hazelnut (Corylus avellana) allergy varies from rather mild oral allergy symptoms to potentially life-threatening anaphylaxis and exhibits geographic and age-related variations. Severity of symptoms depends on the sensitisation profile of the patient and can partially be predicted using 'component-resolved diagnosis'. In our region (young) children predominantly exhibit sensitisation to hazelnut storage proteins Cor a 9 and Cor a 11 that is unrelated to birch pollen allergy and is generally associated with a more severe clinical outcome on consumption on raw and processed hazelnut. In contrast, adults predominantly present with an oral allergy syndrome due to an extensive cross-reactivity between the labile Cor a 1.04 and Bet v 1, the major allergen from birch (Betula verrucosa) pollen. In the absence of a cure, avoidance remains the key measure of effective management, particularly in those patients presenting with a severe form.

Age-dependent sensitization to the 7S-vicilin-like protein Cor a 11 from hazelnut (Corylus avellana) in a birch-endemic region.

BACKGROUND: Hazelnut (Corylus avellana) allergy exhibits age and geographically distinct sensitization patterns that have not yet been fully resolved. OBJECTIVE: To study sensitization to Cor a 11 in different age groups of hazelnut-allergic patients and infants with atopic dermatitis (AD) sensitized to hazelnut in a birch-endemic region. METHODS: Sera from 80 hazelnut-allergic patients, 33 infants under 1 year of age with AD (24 sensitized and 9 not sensitized to hazelnut), 32 healthy control individuals, and 29 birch pollen-allergic but hazelnut-tolerant individuals were tested for immunoglobulin (Ig) E reactivity to Cor a 11 by ImmunoCAP. IgE reactivity to Cor a 1.01, Cor a 1.04, Cor a 8, and Cor a 9 was studied by ISAC microarray. RESULTS: Forty patients (22 preschool children, 10 schoolchildren, and 8 adults) with systemic reactions on consumption of hazelnut were sensitized to Cor a 11 (respective rates of 36%, 40%, and 12.5%). Forty patients (6 preschool children, 10 schoolchildren, and 24 adults) reported oral allergy syndrome but only 2 of them (of preschool age) were sensitized to Cor a 11. Two (8%) of the AD infants sensitized to hazelnut showed IgE reactivity to Cor a 11. This reactivity was not observed in any of the AD infants without sensitization to hazelnut, in any of the birch-pollen allergic patients without hazelnut allergy, or in any of the healthy control individuals. CONCLUSION: Sensitization to Cor a 11 in a birch-endemic region is predominantly found in children with severe hazelnut allergy, a finding that is consistent with observations concerning sensitization to Cor a 9.

Human basophils: a unique biological instrument to detect the allergenicity of food.

BACKGROUND: Labeling of major food allergens is mandatory for the safety of allergic consumers. Although enzyme-linked immunosorbent assay, polymerase chain reaction, and mass spectrometry are sensitive and specific instruments to detect trace amounts of food proteins, they cannot measure the ability of food constituents to trigger activation of mast cells or basophils. AIM: We evaluated the basophil activation test as an instrument to determine the allergenic potential of trace amounts of food allergens in complex matrices. Peanut (Arachis hypogaea) allergy was selected as a proof-of-concept model. METHODS: The study population comprised 5 severely peanut-allergic patients (3 males/2 females; median age, 12 years) all sensitized to 3 major peanut allergens (Ara h 1, Ara h 2, and Ara h 3) and 5 peanut-tolerant individuals (2 males/3 females; median age, 8 years). Basophils from patients and controls were stimulated with pure peanut extract and blank and peanut-spiked (0.1, 0.01, and 0.001 ppm) biscuits (baking time 11, 16, 21, 26 minutes) and chocolate extracts. RESULTS: Blank biscuits and chocolate did not induce cell activation in patients or controls. A comparison between patients and controls showed significantly higher activation of basophils after stimulation with 0.1 and 0.01 ppm of peanut-spiked biscuit at all baking times and peanut-spiked chocolate (P < .05). CONCLUSIONS: The basophil activation test is a highly sensitive and specific tool to detect traces of functionally active food allergens. For biscuits, its accuracy seems independent of baking time. Furthermore, it allows even the most sensitive patients to be included in study protocols.

Age-related sensitization profiles for hazelnut (Corylus avellana) in a birch-endemic region.

BACKGROUND: Symptoms of hazelnut allergy seem related to geographic and possibly age variations in allergen recognition. OBJECTIVE: To investigate sensitization profiles of hazelnut allergy in different age groups in a birch-endemic region using component resolved diagnosis (CRD) by microarray. METHODS: Sixty-five patients with hazelnut allergy, 27 healthy control individuals tolerant to hazelnut, and 34 birch pollen allergic but hazelnut tolerant individuals were included. All blood samples were analyzed using ISAC microarray. RESULTS: Twenty-nine patients with hazelnut allergy suffered from a systemic reaction (17 preschool children with a median age of 2 years, six school children, and six adults), whereas 36 patients reported an oral allergy syndrome (OAS; three preschool and nine school children and 24 adults). In the hazelnut allergic preschool children with systemic reactions, 65% were sensitized to Cor a 9, 12% to Cor a 8, 18% to Cor a 1.04, 6% to Cor a 1.0101, and 29% to Bet v 1. Of the school-aged systemic reactors, 50% were sensitized to Cor a 9, 17% to Cor a 8, 50% to Cor a 1.04 and Cor a 1.0101, and 67% to Bet v 1. In adults with hazelnut allergy, 3.3% were sensitized to Cor a 9, 6.7% to Cor a 8, 90% to Cor a 1.04 and Bet v 1, and 87% to Cor a 1.0101. In regard to systemic reactors in this group, 17% were sensitized to Cor a 9, 33% to Cor a 8 and Cor a 1.0101, and 50% to Cor a 1.04 and Bet v 1. In the patients with OAS, irrespective the age group, all were sensitized to Bet v 1 and over 97% to Cor a 1.04 and Cor a 1.0101. No sensitization to Cor a 9 or Cor a 8 was found in patients with only an OAS. Of the patients with birch pollen allergy, tolerant to hazelnut, none were sensitized to Cor a 9 or Cor a 8, 56% to Cor a 1.0101, 82% to Cor a 1.04, and 92% to Bet v 1. In healthy controls, no sensitization to components of hazelnut, hazel pollen or birch pollen was demonstrable. CONCLUSION: Hazelnut allergy in a birch-endemic region exhibits age-related sensitization profiles with distinct clinical outcomes that can be identified using CRD. The majority of hazelnut allergic preschool and school children in a birch-endemic region show systemic reactions on consumption of processed hazelnut, mostly being sensitized to the hazelnut legumin-like allergen Cor a 9 but unrelated to birch pollen allergy. In contrast, adults generally suffer from an OAS apparently as a result of cross-reactivity between Cor a 1.04 from hazelnut and Bet v 1 from birch pollen.

Basophil activation reveals divergent patient-specific responses to thermally processed peanuts.

INTRODUCTION: The impact of processing on the allergenicity of peanut (Arachis hypogaea) proteins has traditionally been studied using immunoglobulin (Ig) E binding assay. However, as this technique does not assess the potential of an allergen to trigger basophils and mast cells, studies based on it can hardly be considered complete. We evaluated the effect of processing on peanut allergenicity using flow-cytometric quantification of in vitro basophil activation (basophil activation test [BAT]). PATIENTS AND METHODS: Basophils from 10 patients with severe peanut allergy and 3 peanut-tolerant individuals were stimulated with extracts from 5 raw and thermally processed peanut varieties. Data were compared using protein staining (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and IgE immunoblotting. RESULTS: Stimulation with different extracts resulted in patient-dependent and variety-dependent effects on basophil activation. SDS-PAGE revealed a considerable loss of identifiable bands, especially for the South Africa Common Natal, Argentina Runner, and US Virginia varieties. The results of IgE immunoblotting in patients were similar, irrespective of the responses observed in the BAT. CONCLUSIONS: The impact of thermal processing on the capacity of peanuts to trigger basophils seems highly divergent between patients and cannot be predicted using SDS-PAGE or IgE binding. BAT can be considered a complementary tool for the evaluation of food allergenicity.